Ribonucleic acid (RNA) plays an essential role in the translation of the genetic code to produce proteins necessary for cellular function, both in normal cells and neoplastic or diseased cells. In particular, RNA including transfer RNA, messenger RNA or messenger-like RNA, and ribosomal RNA carry and translate the genetic code to sites of protein production. Further, double-stranded RNA and species therefrom, including small inhibitory RNA such as siRNA (short interfering siRNA) and micro RNA or miRNA, play an important role in silencing genetic expression. Other RNA species are found within ribonucleoproteins. For example, telomerase RNA is a critical component of telomerase, an important ribonucleoprotein highly expressed in most cancers. The pathogenesis and regulation of cancer is thus dependent upon RNA-mediated translation and/or inhibitory control of specific genetic code, said genetic code often reflecting mutational events or other alterations within deoxyribonucleic acid (DNA), including epigentic alterations such as hypermethylation, microsatellite alterations, loss of heterozygosity, translocations including inversions and gene fusions, deletions, and point mutations. Further, other RNA species and their associated proteins, although not necessarily being directly involved in neoplastic pathogenesis or regulation, may provide recognizable characterization of neoplasia or disease by being inappropriately expressed or elevated. Such overexpression of RNA thus can delineate cancer or other disease. Recognition of the presence or overexpression of specific RNA, including both coding and non-coding RNA, can enable identification, detection, inference, monitoring, or evaluation of any neoplasm, whether benign, malignant, or premalignant, in humans and animals.
U.S. Pat. No. 6,329,179 B1, incorporated herein in its entirety, teaches that both tumor-associated and non-tumor associated RNA are detectable in plasma and serum. Total RNA is intended herein to refer to a mixture or collection of heterogeneous RNA species or fragments thereof, and is to be understood in the cancer patient to comprise both tumor-associated and non-tumor-associated RNA and further it will be understood that it can be inclusive of heterogeneous RNA of various RNA species and types, which can include messenger RNA, ribosomal RNA, transfer RNA, micro RNA, short interfering RNA, and mitochondrial RNA, and specific species thereof. RNA comprising heterogeneous RNA, such as total RNA, can be extracted from plasma or serum or other non-cellular bodily fluid fractions, the RNA of interest or its cDNA is amplified qualitatively or quantitatively, and the amplified product of an RNA or cDNA species of interest detected. Subsequent art supports these teachings by demonstrating that extracellular RNA of various RNA species are detectable in bodily fluids, for example in co-owned U.S. Pat. No. 6,607,898; Kopreski et al., 1999, Clin. Cancer Res. 5: 1961-1965; Dasi et al., 2001, Lab. Investigation 81: 767-769; Hasselmann et al., 2001, Oncol. Rep. 8: 115-118; Ng et al., 2002, Clin. Chem. 48: 1212-1217; Chen et al., 2000, Clin. Cancer Res. 6: 3823-3826; Silva et al., 2001, Clin. Cancer Res. 7: 2821-2825; Silva et al., 2001, Oncol. Rep. 8: 693-696; Gal et al., 2001, Ann. NY Acad. Sci. 945: 192-194; Durie et al., 2000, Acta Oncol. 39: 789-796; Fleischhacker et al., 2001, Ann. NY Acad. Sci. 945: 179-188; Miura et al., 2003, Oncology 64: 430-434; Kopreski et al., 2001, Ann. NY Acad. Sci. 945: 172-178; Wong et al., J. Clin. Pathol. 2004, 57: 766-768′ and Ma et al., Haematologica, 2007, 92: 170-175, said references incorporated herein in their entirety. Detection of tumor-associated RNA in plasma or serum or non-cellular bodily fluid fractions thus provides a method for detecting, diagnosing, inferring, evaluating or monitoring cancer or premalignancy in a human or animal. Similarly, detection of extracellular RNA in bodily fluids enables the evaluation and monitoring of treatments and therapies for cancer and other diseases.
Neoplasia is characterized by varying degrees of invasiveness, metastatic potential, and resistance or responsiveness to particular therapies. Furthermore, these characteristics for a given neoplasia may change over time, for example by becoming progressively more malignant, invasive, metastatic, heterogeneous, undifferentiated, or treatment-resistant. Phenotypic changes often reflect underlying molecular changes. In particular, the relative ratio of particular RNA species, including coding and non-coding species, to each other, and/or to DNA, and/or to proteins can determine the characteristics of the neoplasia, and further enable the diagnosis, detection, evaluation, or monitoring of cancer and premalignancy. One group of tumor-associated RNA expressed or over-expressed in neoplastic disease are RNA associated with DNA translocations, including herein inversions and gene fusions. The presence of a specific translocation or gene fusion may further characterize a neoplastic disease.
Analysis in an absolute or relative fashion of extracellular RNA species to each other, and/or to extracellular DNA, and/or to extracellular protein, would thus be useful as a method for detecting, diagnosing, inferring, characterizing, or monitoring cancer or premalignancy in a human or animal. Said analysis further enables the selection and monitoring of treatment. Cancer treatments can target or inhibit specific gene products or gene pathways, such as for example but not limitation, fusion gene targets ore receptor tyrosine kinase targets. Cancer treatments can further target or inhibit specific mRNA targets, for example using anti-sense RNA or using siRNA therapies.
Thus, there is a need in the art for methods of comparing the amount or concentration or relative ratio of two or more plasma or serum RNA species or fragments thereof to permit diagnosis, detection, inference, evaluation, or monitoring of neoplastic disease in a human or animal. It is to be explicitly understood that said comparison of two or more RNA species may include comparison of non-mutated tumor RNA to tumor RNA; tumor RNA to non-mutated non-tumor RNA; coding RNA to coding RNA; coding RNA to non-coding RNA; and non-coding RNA to non-coding RNA; or any combination thereof. Further more a RNA may be compared to a DNA.
Furthermore, there is a need for methods of comparing the amount or concentration or ratio of one or more extracellular RNA species to the amount or concentration of total RNA or extracellular DNA or protein present in the plasma, serum, or bodily fluid of a human or animal for the diagnosing, detecting, inferring, evaluating, or monitoring cancer and other neoplastic diseases in the human or animal.
It is further understood that the present invention provides methods for detecting RNA associated with translocated DNA and gene fusions, which are often associated with neoplastic disease. Said RNA may be detected in a qualitative or quantitative fashion either itself, or in comparison to a reference RNA, or in combination with other tumor-associated RNA and/or tumor-associated DNA.